Purification and Identification of a Novel Angiotensin Converting Enzyme Inhibitory Peptide from the Enzymatic Hydrolysate of Lepidotrigla microptera.

In this study, Lepidotrigla microptera were hydrolyzed with four different proteolytic enzymes (Papain, neutrase, flavourzyme, and alcalase), and their distribution of molecular weights and ACE-inhibitory activity were tested. The alcalase hydrolysates showed the maximum ACE-inhibitory activity. A novel ACE-inhibitory peptide was isolated and purified from Lepidotrigla microptera protein hydrolysate (LMPH) using ultrafiltration, gel filtration chromatography, and preparative high performance liquid chromatography (prep-HPLC). The amino acid sequence of the purified peptide was identified as Phe-Leu-Thr-Ala-Gly-Leu-Leu-Asp (DLTAGLLE), and the IC50 value was 0.13 mg/mL. The ACE-inhibitory activity of DLTAGLLE was stable across a range of temperatures (<100 °C) and pH values (3.0−11.0) and retained after gastrointestinal digestion. DLTAGLLE was further identified as a noncompetitive inhibitor by Lineweaver−Burk plot. The molecular docking simulation showed that DLTAGLLE showed a high binding affinity with ACE sites by seven short hydrogen bonds. As the first reported antihypertensive peptide extracted from alcalase hydrolysate of Lepidotrigla microptera, DLTAGLLE has the potential to develop functional food or novel ACE-inhibitor drugs.

A universal random DNA amplification and labeling strategy for microarray to detect multiple pathogens of aquatic animals.

Diseases caused by bacteria, fungi, and viruses pose a great threat to aquaculture. As DNA microarrays can be used to detect multiple pathogens, here we reported an array with the potential to simultaneously detect 13 bacterial and 11 viral pathogens of aquatic animals. The array included 853 oligonucleotide probes (20-40 mer) complementary to various virus-specific sequences and four chromosomal loci (16S rRNA, gyrB, dnaJ, and recA) of bacteria. Multiplex PCR, phi29 DNA polymerase, and a Klenow fragment-based method were evaluated for amplifying and labeling the nucleic acid of pathogens. While array hybridization signals were most intense using pathogen sequences amplified by multiplex PCR, the phi29 DNA polymerase method was more convenient and ideal since it did not require sequence-specific primers that could bias against detecting novel pathogens. The feasibility of the phi29 DNA polymerase-based microarray strategy was also demonstrated by detecting multiple unknown pathogens from four samples of diseased fish and shrimps.

A graphene tip coupled with liquid chromatography tandem mass spectrometry for the determination of four synthetic adulterants in slimming supplements.

Slimming supplements were popularly sold online driven by the increasement of obesity and the development of social networking platform. However, events of drug abuse in slimming supplements were also frequently reported. In this study, a graphene tip solid-phase extraction (Gtip SPE) and ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method was established for determining fenfluramine, phenolphthalein, bumetanide, and sibutramine in slimming supplements. It was validated in terms of linearity (0.9985-0.9995), LOD (1.8ngmL-1), LOQ (5.6ngmL-1), intra-day precision (<5.1%), inter-day precision (<7.3%), and recovery (82.9-95.2%). Sibutramine is the most commonly used drug, which was detected in Bihais, Galong, and Aolist, with content 12.4, 3.6, 20.3mgg-1, respectively. Phenolphthalein was also found with content lower than 5.2mgg-1. The successful application of Gtip SPE and UPLC-MS/MS method indicated its advantage in analyzing low level of contaminates resulted from violation of regulation.

Zwitterionic hydrophilic interaction solid-phase extraction and multi-dimensional mass spectrometry for shotgun lipidomic study of Hypophthalmichthys nobilis.

Zwitterionic hydrophilic interaction liquid chromatography (ZIC-HILIC) material was used as solid-phase extraction sorbent for purification of phospholipids from Hypophthalmichthys nobilis. The conditions were optimized to be pH 6, flow rate 2.0mL·min(-1), loading breakthrough volume ⩽5mL, and eluting solvent 5mL. Afterwards, the extracts were analyzed by multi-dimensional mass spectrometry (MDMS) based shotgun lipidomics; 20 species of phosphatidylcholine (PC), 22 species of phosphatidylethanoamine (PE), 15 species of phosphatidylserine (PS), and 5 species of phosphatidylinositol (PI) were identified, with content 224.1, 124.1, 27.4, and 34.7µg·g(-1), respectively. The MDMS method was validated in terms of linearity (0.9963-0.9988), LOD (3.7ng·mL(-1)), LOQ (9.8ng·mL(-1)), intra-day precision (<3.64%), inter-day precision (<5.31%), and recovery (78.8-85.6%). ZIC-HILIC and MDMS shotgun lipidomics are efficient for studying phospholipids in H. nobilis.

Analysis of trace levels of sulfonamides in fish tissue using micro-scale pipette tip-matrix solid-phase dispersion and fast liquid chromatography tandem mass spectrometry.

A micro-scale matrix solid-phase dispersion (MSPD) technique, using hydrophilic-lipophilic balance (HLB) material as sorbent and a pipette tip (PT) as the cartridge, was developed for the extraction and purification of sulfonamides in fish tissue. Eluates from PT-MSPD were analyzed using fast liquid chromatography and tandem mass spectrometry (LC-MS/MS). The method was fully validated; good linearity was obtained with correlation coefficients greater than 0.99. Precision and accuracy (RSD%) were in the range 1.4-10.3% while mean recoveries were 70.6-95.5%. With this technique, 15 aquatic samples (Collichthys niveatus) were analyzed for sulfonamides. The whole procedure took only 13min (5min for PT-MSPD and 8min for LC), materials for each sample included 5.1mL solvents (0.3mL for PT-MSPD and 4.8mL for LC), and 20mg HLB sorbent. Generally speaking, this method is indeed practical and particularly suitable for widespread drug residue analysis.

Enzyme-linked immunosorbent assay based on a monoclonal antibody for the detection of the insecticide triazophos: assay optimization and application to environmental samples.

A direct competitive enzyme-linked immunosorbent assay (ELISA) for triazophos was developed, which was based on the THHe monoclonal antibody (McAb) and a heterologous enzyme tracer (THBu-HRP). The influence of several physicochemical factors (temperature, time, pH, salt, detergent, and solvent) on the immunoassay was studied. For the standard curve, an I50 of 0.21 microg/L and a limit of detection (I20) of 0.02 microg/L was obtained in a high salt concentration buffer (0.05 M PBS, pH 6.0) with 0.05% BSA, which means an almost 3-fold improvement in the assay sensitivity in comparison with the nonoptimized conditions. The optimized ELISA has been used to quantify triazophos in water and soil samples spiked at different amounts. The excellent recoveries achieved confirmed the potential of the immunoassay for environmental monitoring of triazophos in waters and soils without purification steps.